Abstract The goal of this proposal is to provide a foundation to prepare the applicant, Dr. Tim Heden, for a career as an independent scientist. Dr. Heden has previously undergone Ph.D. training in the field of nutrition and exercise physiology and has completed his first year as a postdoctoral trainee studying phospholipid metabolism. The proposed training plan will enhance the applicant?s scientific skills and better equip him to reach his long-term goal of becoming an independent scientist. The proposed research will identify the role of skeletal muscle mitochondrial phosphatidylethanolamine (PE) biosynthesis in respiratory capacity and substrate metabolism in skeletal muscle. PE is synthesized by phosphatidylserine decarboxylase (PSD) that resides in the inner- membrane of mitochondria. Preliminary data in mice show that exercise training increased skeletal muscle mitochondrial PE content and PSD expression, whereas an 8-wk HFD reduced mitochondrial PE content without changing PSD expression. These findings suggest mitochondrial PE may mediate the differential metabolic phenotype between muscles from exercise-trained and HFD-fed mice. To better understand the biological function of skeletal muscle PSD, we performed preliminary mechanistic experiments to examine the effects of PSD knockdown in C2C12 myotubes. A lentivirus-mediated knockdown of PSD in these myotubes reduced mitochondrial PE content, maximal respiration, fatty acid oxidation, and complex II activity, but also increased fission, mitophagy, and glucose utilization. These data suggest that mitochondrial PE biosynthesis directly regulates skeletal muscle mitochondrial respiratory capacity and substrate metabolism. To gain better insight into the role of skeletal muscle mitochondrial PE biosynthesis in vivo, we generated mice with skeletal muscle- specific knockout of PSD (PSD-MKO). In this proposal, we will test the hypothesis that skeletal muscle mitochondrial PE modulates cellular respiration, and its absence results in mice that have low aerobic capacity and are more prone to developing insulin resistance. Data from these studies will provide fundamental insights into the regulation of skeletal muscle respiration and substrate metabolism and introduce mitochondrial PE as a potential key player in metabolic disease development.